A nitrile hydratase is an enzyme having nitrile hydration activity that catalyses the hydration of a nitrile group to an amide group. Also, corresponding amide compounds can be produced from nitrile compounds by using the enzyme or a microbial cell or the like containing the enzyme. Compared with conventional chemical synthetic methods, this method is known to have high conversion rate and high selectivity rate from a nitrile compound to a corresponding amide compound.
Examples of microorganisms that produce a nitrile hydratase include the genus Corynebacterium, genus Pseudomonas, genus Rhodococcus, genus Rhizobium, genus Klebsiella, genus Pseudonocardia and the like. Among those, Rhodococcus rhodochrous J1 strain has been used for industrial production of acrylamides, and its usefulness has been verified. Furthermore, a gene encoding a nitrile hydratase produced by the strain has been identified (see Patent Publication 1).
Meanwhile, introducing a mutation into a nitrile hydratase has been attempted not only to use a nitrile hydratase isolated from a naturally existing microorganism or its gene, but also to change its activity, substrate specificity, Vmax, Km, heat stability, stability against a substrate, stability against a subsequent product and the like of a nitrile hydratase. Regarding the nitrile hydratase in Pseudonocardia thermophila JCM 3095, from its three dimensional structure data, presumed sites relating to the substrate specificity or thermal stability are obtained, and mutant enzymes with modified substrate specificity were obtained among them (see Patent Publications 2 to 4). Also, nitrile hydratase genes with improved heat resistance and amide-compound resistance have been produced by the inventors of the invention (see Patent Publications 5 to 9).
However, developing a nitrile hydratase which has further enhanced heat resistance and resistance to amide compounds and can react at high temperatures and using the nitrile hydratase for production of an amide compound are very useful from the viewpoint of production cost like cost involved with catalyst, and obtaining enzymes with such performance is especially desired so as to achieve a reduction in the enzyme amount for reactions and in production costs or the like.